Identiﬁcation and Characterization of a New Microalga Dysmorphococcus globosus -HI from the Himalayan Region as a Potential Source of Natural Astaxanthin

Simple Summary: The natural astaxanthin from algae (marketed only 5%) is the most powerful antioxidant for health compared to the synthetic that shares about 95% of the market. Therefore, there is a huge demand for natural astaxanthin from algae, which can accumulate signiﬁcantly higher astaxanthin. We have isolated a new algal strain from the Himalayan region, Northern India, which was identiﬁed as Dysmorphococcus globosus -HI based on morphological and molecular analysis. in the laboratory. Among different tested media, MBBM and 3N-BBM provided the maximum growth and astaxanthin production. The highest biomass production (1.14 g L − 1 ) was observed in the modiﬁed BBM medium. It is an excellent source of producing natural astaxanthin, to a tune of 391 mg L − 1 , which is greater than any other known algal species. The productivity of astaxanthin was about 15.6 mg L − 1 d − 1 under normal conditions, which is higher than the commercially used H. pluvialis species. It is the ﬁrst report of natural astaxanthin production from D. globosus -HI that has great potential for commercial application. Abstract: Synthesized astaxanthin (ASX), stereoisomers of 3S,3 (cid:48) R, 3R,3 (cid:48) R, and 3S,3 (cid:48) S, have over 95% market share and have relatively poor antioxidant and bioactivity properties, with persistent issues in terms of biological functions, health beneﬁts, and biosafety if compared to natural ASX. Bioprospect-ing of new microalgal strains could be vital for a new source of powerful antioxidant (ASX). In this study, a new algal strain was isolated from the Indian foothills of the Himalayas. Its identity was discerned by morphological and DNA barcode studies. It is a unicellular spheroidal cell-shaped alga with 100–200 µ m diameter. The isolate has 93.4% similarity to Dysmorphococcus globosus species based on 18S-rDNA phylogenetic analysis and named as D. globosus -HI (HI stands for Himalayan India). Its growth and major cellular components (carotenoids, carbohydrates, protein, lipids, fatty acid proﬁle, and ASX) were optimized using the seven different culture media. The highest biomass (1.14 g L − 1 ) was observed in the MBBM medium, with a speciﬁc growth rate (0.087 day − 1 ), division/day (0.125), and cellular yield (6.16 × 10 6 cells/mL). The highest carotenoids (1.56 mg g − 1 ), lipids (32.5 mg L − 1 ), and carbohydrates (135.62 mg L − 1 ) were recorded in the 3N-BBM medium. The maximum ω 3-FAs (17.78%), ω 6-FAs (23.11%), and ω 9-FAs (7.06%) were observed in MBBM, JW, and BG-11 medium respectively. The highest amount of antioxidant ASX was accumulated in the 3N-BBM medium (391 mg L − 1 ). It is more than any other known algal species used in the production of natural ASX. The optimized biochemical studies on the D. globosus -HI strain should fulﬁll the increasing demand for natural ASX for commercial application.

Among carotenoids, astaxanthin (ASX), a 3,3 -dihydroxylated and 4,4 -diketolated derivative of β-carotene (3,3 -hydroxy-β,β-carotene-4,4 -dione), is one of the most valuable bioproducts due to its high antioxidant capacity [31,32]. The ASX has the most positive antioxidation capacity due to the α-hydroxy ketone functional groups, which involved the singlet oxygen-quenching, reduction of low-density lipoprotein, anticancer activities, and enhancement of immune responses [33,34]. It has been industrially exploited as a feed dye, particularly as a feed supplement in aquaculture and poultry farming [3,6,7]. Due to the exceptional antioxidant capacity of ASX, the trend of its commercial uses in pharmaceutical, nutraceutical, and medical applications is expected to be dramatically increased in the future [25,26].
It is estimated that by 2025 the worldwide global market of ASX shall reach US$ 2.57 billion per year [35]. Currently, a small amount (below 5%) of natural ASX is produced worldwide from freshwater alga Haematococcus pluvialis with an approximate cost of US$2500-7000/kg [28,36,37]; the rest about 95% of ASX marketed is chemically synthesized from the petrochemical resources [38,39], comparatively at low cost $1000/kg [40]. However, synthesized ASX has persistent issues in terms of biological functions, health benefits, and biosafety [41]. The synthesized ASX contaminated with other chemical byproducts/intermediates may also have adverse impacts on human health when consumed through supplemented foods and pharmaceuticals [4,38]. Moreover, synthesized ASX has a mixture of (3S,3 R), (3R,3 R), and (3S,3 S) stereoisomers with a proportion ratio of 1:2:1, respectively [26,31], making it an antioxidant with poor properties than natural ones [41]. The natural ASX has two stereoisomers, (3S,3 S)and (3R,3 R), which are responsible for its greater bioactivity and higher antioxidant properties [26,31,41]. Different biological approaches have been implemented for obtaining ASX from natural resources [38], like direct extraction from marine animals and crustacean waste (krill, shrimp, and crab) [7,33]. The ASX extracted from marine animals/crustacean waste is not suitable for hygienic purposes due to marine pollution, accumulated heavy metals, residual antibiotics, and microplastic contamination in marine animals [11]. Moreover, ASX from marine animals/crustacean waste is low in productivity and includes high extraction costs, thus limiting its commercial-scale production [38]. Despite high production costs, microalgae-based ASX may be advantageous in terms of sustainability, safety control, productivity, and production area [11,38].
Besides Haematococcus pluvialis (standard ASX-producing algal strains) [12,42,43], other microalgae species are also used to produce a natural ASX, like Chlorella zofingiensis [44], Chlorella protothecoides [45], Scenedesmus sp. [46] and Neochloris wimmeri [47] with a low amount of ASX (1.1 to 10.72 mg L −1 ). Therefore, there is great need to isolate and identify novel algal strains that can sustain high growth rates and accumulate higher ASX. The ASX productivity is greatly affected by altering the growth conditions, i.e., the culture Biology 2022, 11, 884 3 of 25 media [48,49]. Thus, ASX productivity can be improved via media engineering and by thereby improving the growth of an ASX-producing strain. Thus, identifying the best culture medium is vital for attaining the highest ASX yield [19,50].
In the present study, we have isolated a new algal strain from the foothills of the Himalayan region, Northern India. This organism gradually turns to orange-red from the green stage during prolonged cultivation, a similar phenomenon is reported for H. pluvialis, indicating its importance as a new source of ASX production. The genetic identification and the morphological and biological properties of the isolated microalga were investigated. In addition to the growth characteristics, the composition of the major cellular components, i.e., carbohydrates, protein, lipids, fatty acid profile, and ASX were determined. We aimed to identify the best medium that could be useful in attaining the highest ASX accumulation in the isolated strain. Thus, seven culture media were studied to optimize the strain growth for higher ASX production. The strain was identified as D. globosus-HI strain from the Himalayan region, Northern India. It is the first report that highlights its potential for natural ASX production at the par of H. pluvialis or more under optimized conditions.

Sample Collection, Isolation, and Growth Conditions
A natural algal strain was isolated from the foothills, Himachal Pradesh, India (latitude 32 • 6 37.9512 N and longitude 76 • 32 10.4064 E). The collected algal samples were enriched using Bold's Basal Medium (BBM) (Table S1) by adding ten mL of the collected samples to 50 mL BBM liquid medium in Erlenmeyer flasks (100 mL). A purified culture (axenic culture) was obtained by subjecting the samples to successive rounds of streaking across BBM plates containing antibiotics, i.e., Ampicillin (100 µg mL −1 ) and Cefotaxime (50 µg mL −1 ), and were maintained in axenic conditions by adding one mL of antibiotic into one L of BBM culture medium. The purified algal culture was maintained in the liquid medium, incubated at 25 ± 2 • C, with continuous illumination of light (100 µmolm −2 s −1 ), and was kept under constant shaking on an orbital shaker at 150 rpm.

Molecular Identification and Phylogenetic Analysis
The molecular identification of the isolated strain was conducted using an 18S rDNA sequencing method. To perform the polymerase chain reaction (PCR), total genomic DNA was extracted from 400 mg of wet biomass culture during the exponential phase following the CTAB method [51]. The conserved DNA region corresponding to 18S rDNA was amplified using the 18S primers for the PCR amplification, forward NS1-5 -GTAGTCATATGCTTGTCTC-3 and reverse NS6-5 -GCATCACAGACCTGTTAT TGCCTC-3 . The PCR amplification was carried out in a 20 µL reaction containing 10 mM dNTP, 5 pmol of each primer, 10× Taq buffer, 0.5 U of Taq polymerase (Real Biotech Corporation, Delhi, India), and 100 ng template DNA. The conditions for PCR amplification were an initial denaturation for 3 min at 95 • C, followed by 35 cycles of denaturation for 30 s at 95 • C, primer annealing at 64 • C for 30 s, and extension for 30 s at 72 • C. The PCR products were separated by Electrophoresis, using 1.5% agarose gel, and visualized in the Bio-Rad Gel doc. system. The size of the amplified DNA "PCR product" using the 18S primers was between 1000 bp and 1500 bp. The DNA sequencing was quantified by NanoDrop (Thermo Fisher one/one C Microvolume UV-Vis Spectrophotometer) and was carried out by Macrogen Bioservices Inc. Seoul, Korea, and checked for similarity against other publicly available sequences using the Basic Local Alignment Search Tool (BLAST) algorithm to identify homologous taxa available on the NCBI DNA database. The DNA sequences were aligned automatically using the ClustalW alignment algorithm under default parameters using Molecular Genetics Analysis (MEGA X) software [52]. A phylogenetic tree was constructed using the maximum-likelihood algorithms method under default parameters [53] using MEGA X software [54]. The evolutionary distances were computed using the p-distance method [55] and were in the units of the number of

Morphological Characterization of Isolate by Light Microscopy
The isolated microalga was examined under a light microscope (LM, Eclipse 80i; Nikon Co., Tokyo, Japan). Images were obtained using a camera (DXM 1200C; Nikon Co., Tokyo, Japan), and the cell size and shape were calculated with an image analyzer (NIS-Elements BR 3.0; Nikon Co., Tokyo, Japan). The cell and shape, chloroplast color and shape, number of pyrenoids, and presence or absence of flagella were recorded.

Assessing Different Growth Media for Optimum Growth
The isolated strain was grown experimentally in 7 common freshwater media (using three replicates), namely BBM [57], MBBM [58], 3N-BBM [59], BG-11 [60], OHM [61], CM Medium, modified [62] and JW (Jaworski's Medium) [63]. The culture media composition (g L −1 ) and chemical characteristics of the 7 culture media are presented in Table S1. The 7 culture media were individually prepared in autoclaved Erlenmeyer flasks (1 L) with a working volume of 700 mL and reagents of an analytical grade. To avoid nutrient interferences during subculturing, the supernatant of the old medium was discarded, and the starting inoculum was used at the 0.1 OD 750 for each subculture using a fresh medium. The pH for all the examined media was maintained at 6.9-7.2. The experiment was carried out under a 16:8 light/dark cycle with cool white fluorescent lamps by the side of the flasks providing 100 µmolm −2 s −1 and constant shaking on an orbital shaker at 150 rpm. When the stock culture reached the early stationary phase (25 days after cultivation), the cells were collected via centrifugation at 6000 rpm for 5 min and biomass (cell dry weight; CDW) and some biochemical parameters were determined as protein and carbohydrates, lipid content, fatty acids, total carotenoids, and ASX content and productivity. The optical density (OD) of the culture was measured every two days at 750 nm [64] using a UV-1800 Spectrophotometer (Shimadzu, Japan). The dry biomass production (g L −1 ) was quantified twice using dry cell weight and the filtration process. Culture samples (20 mL) were filtered using a pre-weighted Macherey-Nagel GF-1 glass-fiber filter and were dried at 60 • C overnight, then the biomass productivity (g L −1 d −1 ) was calculated according to Rizwan et al. [65] using the following Equation (1): where X f and X i correspond to final and initial CDW (g L −1 ), at the final time CDW (t f ) and initial time (t i ), respectively. The initial CDW was measured directly after inoculation of the culture, while the final CDW was after 25 days of cultivation (during the early stationary phase). The microalgae specific growth rate (µ; d −1 ), doubling time, divisions per day, and maximum cellular yield were estimated according to Guillard [66] and Wood et al. [67] using the following Equations (2)-(5): Divisions per day = µ 0.6931 (4)  Maximum cellular yield = N f − N i (5) where N f and N i correspond to the final and initial cell number (cell mL −1 ), respectively, related to their specific final (t f ) and initial time (t i ) in days.

Determination of Chlorophyll a and b and Total Carotenoid Content
The total chlorophyll a and b and total carotenoid contents were determined according to Lichtenthaler [68]. One mL aliquot culture was centrifuged, and the pellets were collected while the supernatant was discarded. One mL of methanol (99%) was added to the collected pellets and the samples were stored at 4 • C overnight under dark conditions. Subsequently, samples were centrifuged, and chlorophylls a and b and carotenoid contents (µg mL −1 ) were estimated by measuring the optical absorbance of the supernatant at 470, 652, and 665 nm using Spectramax M3 multimode spectrophotometer (Molecular Devices, San Jose, CA, USA) and expressed as µg/mL.

Measurement of Protein and Carbohydrates
At the late exponential phase, the total algal biomass was harvested by centrifugation at 6000 rpm for 5 min and dried at 60 • C overnight, then the dried biomass was used for protein and carbohydrates measurements. Protein content was determined following the Lowry method using bovine serum albumin as a standard [69]. Total carbohydrate content was determined following the phenol-sulfuric method and was quantified by a standard glucose curve at 490 nm using Spectramax M3 multimode spectrophotometer (Molecular Devices, San Jose, CA, USA) [70]. Protein and total carbohydrate contents were expressed as mg L −1 .

Total Lipid Content and Fatty Acids (FAs) Profile Analysis
The total lipids were extracted from the dried algal biomass following the method of Bligh and Dyer [71]. In a glass vial, 500 mg of ground-dried algal biomass (overnight in the oven at 55-60 • C) was mixed with 3 mL of 2:1 chloroform/methanol mixture, then 0.9 mL of distilled water was added and kept overnight on a shaker. After overnight shaking, an additional one mL chloroform and 0.9 mL of distilled water were added, shaken for 4 h and the lipid layers were allowed to separate. The bottom chloroform layer was aspirated and filtered through Whatman No. 1 filter paper into a pre-weighted glass vial. Chloroform was evaporated using a rotary evaporator (BUCHI, Rotavapor II) at 40 • C and the total lipid (mg g −1 ) was calculated by subtracting the pre-weighed glass vial from that of the vial containing the lipid. The lipid productivity (mg g −1 day −1 ) was calculated by dividing the lipid content (mg g −1 ) by the number of days "from cultivation to harvest".
As for the FAs profile analysis, first, the lipid trans-esterification was undertaken by mixing ten mg of the extracted lipids with one mL of hexane (99%) in a test tube, then 200 µL of methanolic KOH (2 M) was used as a catalyst [72] and the mixture was vigorously agitated using a vortexer for 5 min. Then, the upper clear supernatant (hexane) was collected for FAs profile analysis. The quantification of FAs profile analysis was carried out using a gas chromatograph-FID (Agilent GC) equipped with an OmegaWax 250 capillary column (30 m length × 0.25 mm internal diameter × 0.25 µm phase thickness, SUPELCO) and a flame ionization detector. The operating conditions were as follows: a split ratio of 1:10, injection volume of 1 µL, Nitrogen carrier gas with a constant linear velocity of 33.9 cm s −1, and pumping rate of 40-, 400-, and 30-mL min −1 for H 2 , air and makeup gas (Nitrogen), respectively. The injector temperature was 270 • C and the detector temperature was 280 • C, whereas the oven temperature started at 140 • C (for 5 min) and then increased at the rate of 4 • C min −1 to reach 240 • C, and was then maintained for 20 min. The Heptadecanoic acid (C17:0) was used as an internal standard. Fatty acids were identified by comparing the samples retention times with appropriate FAME standards (Supelco standard FAME mixture-; Sigma Chemical Company, St. Louis, MI, USA) and the data for each individual components are expressed as a percentage of the total content [72].

Astaxanthin Analysis and Quantification
The analytical thin-layer chromatography (TLC analysis) was used for the detection of the pigments and ASX in the isolated algal strain. After successful detection, the HPLC was used for efficient quantification of ASX via their retention values.

Thin Layer Chromatography (TLC)
A TLC-pre-coated silica gel H60 thin layer chromatography plate (20 cm × 20 cm Merck, Darmstadt, Germany) was used for the separation of different pigments according to Kobayashi et al. [73] and Chekanov et al. [74] At room temperature, a known weight (15 mg) of the different algal samples was extracted in 2 mL acetone (Sigma-Aldrich Chemical Co., St. Louis, MI, USA), and vortexed with a glass bead for 4 min, centrifuged for 5 min, and this process was repeated until the cell debris became colorless. The algal pigment extracts were dried under N2 gas flow under low light conditions. The samples were resuspended in the petroleum ether, and were then carefully spotted (20 µL of the different samples) to a pre-heated silica Gel chromatography plate, with the Standards (B-carotene and ASX, with a concentration of one gm mL −1 ; Sigma-Aldrich Co., St. Louis, MI, USA) and were allowed to completely dry. The plate was transferred to a highly saturated chamber with a fresh mixture of organic solvents (acetone and n-hexane at a ratio of 30:70% "v/v") as a mobile phase and was kept for 30 min at room temperature and under low light conditions. They were then removed and quickly marked on the solvent level and the center of the pigment bands with a pencil. The retardation indices (RFs) for each pigment were calculated using the following equation:

RFs =
Distance traveled by each pigment Distance traveled by the solvent (mobile phase) (9) The distance traveled is the distance from the starting point to the geometrical center of the spots corresponding to each pigment or the mobile phase.

High-Performance Liquid Chromatography (HPLC)
The ASX was repeatedly extracted (three times) until the cell debris was colorless by adding ten ml of dichloromethane (DCM) to 50 mg of lyophilized dry biomass until the cell debris became colorless. The ASX-DCM extract was evaporated with the HeidolphHei-VAP rotary evaporator, and was then saponified for 3 h at room temperature (23 • C ± 2) under dark conditions by adding 2.25 mL of acetone, 0.25 mL of methanol and 0.5 mL of NaOH (0.05 M in methanol) [75]. Afterward, petroleum ether (three mL) was added, and the mixture was washed with three mL of 10% aqueous NaCl solution, and was then centrifuged for two min at 5500 rpm. The lower phase was discarded and the washing step with the NaCl solution was repeated twice. The organic phase was evaporated and the extracted ASX was dissolved using three mL of solvent B (methanol, methyl tert-butyl ether (MTBE), water, 8:89:3, v/v) and was filtered with a 0.22 mm disposable nylon syringe filter from Berrytec (Germany). The de-esterified ASX was determined using an HPLC unit (LC-20AB, Shimadzu, Japan) with a diode-array detector (SPD-M20A, Shimadzu, Japan) using a YMC Carotenoid column (C30, 3 mm, 150 × 4.6 mm, YMC Co., Kyoto, Japan). As for the mobile phase, solvent A (methanol, MTBE, water, 81:15:4, v/v) and solvent B (methanol, MTBE, water, 8:89:3, v/v) were used with the following gradient: 2% solvent B for 11 min, a linear gradient from 2% solvent B to 40% solvent B for 7 min, 40% solvent B for 6.5 min followed by a linear gradient to 100% solvent B for 2.5 min, 100% solvent B for 3 min, a linear gradient to 2% solvent B for 3 min, held for 7 min. The flow rate was 1 mL min −1 , the injection volume was 10 µL and the column temperature was maintained at 22 ± 2 • C. For the ASX quantification, a calibration curve was established using standard ASX (Sigma Alderish Company), in which a set of serial dilutions (ranging from 0 to 10 mg L −1 ) of stranded ASX (dissolved in DMSO) were prepared, and then the signal of the diode-array detector was recorded at 478 nm and the corresponding peaks using the HPLC was measured. The limit of detection (LOD) and the limit of quantification (LOQ) were calculated according to the calibration curve [36,76]. The concentration of standard ASX was used as a reference to calculate the concentration of ASX in the sample according to the HPLC peak area. ASX was expressed in mg L −1 and as % (w/w). The ASX productivity (mg L −1 day −1 ) was calculated by dividing the ASX content (mg L −1 ) by the number of days, the "cultivation to harvest".
The HPLC-grade methanol, MTBE, and ASX were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MI, USA). Other chemicals and reagents were purchased from local companies. Deionized water was purified using a Milli-Q water system (Merck Millipore, Billerica, MA, USA) and was filtered through a 0.22 m 2 membrane for HPLC analysis.

Statistical Analysis
The obtained data were subjected to ANOVA using one-way CRD in the Glmmix procedure in SAS 9.4 [77]. Differences between means were separated using Tukey's honestly significant difference HSD test at p ≤ 0.05. All data were collected in triplicates as mentioned in the figures and tables.

Cell Morphology
The morphological characteristics of the isolated microalga were investigated using a light microscope. The isolated strain was a unicellular dark green microalga, some cells were observed in aggregation. These cells grew in variable forms/shapes ranging from globose to spheroidal, with a cell size ranging from 100 to 200 µm ( Figure 1A,F). Interestingly, the color of the isolated strain changed from green to red, indicating the aging of the culture while accumulating pigments. The growth of D. globosus-HI was distinguished based on cells' age, i.e., young green culture (young cells) ( Figure 1A-C), which is characterized by a green color for around 20 days. At the stationary phase, culture without the replacement of a new nutrient medium turned into reddish orange (old cells) ( Figure 1D-F). The green and red cells were also observed on agar plates ( Figure S1A,B) and in liquid media ( Figure S1C,D). Throughout the observations, two ages of the cultures were predominated, and the chloroplast seems to occupy the almost whole-cell lumen ( Figure 1A-F). The D. globosus-HI cell showed the two isokontic flagella (FL and FR), broadly ovoid-shaped lorica (L) around the green individual cell, and an eye spot (ES) or stigma as a thin or streak-like structure ( Figure 2A). The cell wall appears to be hyaline ( Figure 2B,C). Each green cell showed 1-2 pyrenoids (PY) surrounded by a starch sheath and has a single urn-or parietal-shaped chloroplast (CP) ( Figure 2B,C). Green cells in groups were surrounded by the sporangium wall (SW) or the parental lorica ( Figure 2B,C). Evidence for asexual reproduction in D. globosus-HI ( Figure 2D-G) showed the aplanospores, with each mother cell containing 4-16 daughter cells.

Molecular Identification and Phylogenetic Analysis
In the BLAST analysis, 1121 nucleotides of the 18S rDNA-related gene sequences were obtained from the isolated microalgae. This sequence was assigned an accession number of H210309 after submission to the National Center for Biotechnology Information (NCBI). The nucleotide sequences of the other 16 algal species were obtained from

Molecular Identification and Phylogenetic Analysis
In the BLAST analysis, 1121 nucleotides of the 18S rDNA-related gene sequences were obtained from the isolated microalgae. This sequence was assigned an accession number of H210309 after submission to the National Center for Biotechnology Information (NCBI). The nucleotide sequences of the other 16 algal species were obtained from

Molecular Identification and Phylogenetic Analysis
In the BLAST analysis, 1121 nucleotides of the 18S rDNA-related gene sequences were obtained from the isolated microalgae. This sequence was assigned an accession number of H210309 after submission to the National Center for Biotechnology Information Biology 2022, 11, 884 9 of 25 (NCBI). The nucleotide sequences of the other 16 algal species were obtained from the NCBI database based on the BLAST results of accession number H210309 for comparative alignment analyses of 18S rDNA. The BLAST analysis revealed that our isolate was highly similar to Dysmorphococcus globosus as per the NCBI database. The Dysmorphococcus globosus SAG 20-1 (KM020136.1) and D. globosus (X91629.1) species shared the highest similarities (93.14 to 93.45%) to our isolated microalga.
The phylogenetic analysis was performed on the basis of blast results (Figure 3) for the maximum likelihood method and involved 16 nucleotide sequences using MEGA X software [54]. All ambiguous positions were removed for each sequence pair (pairwise deletion option) and a total of 2879 positions in the final dataset were observed. The resultant tree using the 18S rDNA genes revealed that the newly isolated microalga strain has a maximum DNA sequence similarity with Dysmorphococcus globosus strain SAG 20-1 (KM020136.1) and D. globosus (X91629.1) (Figure 3). Based on the phylogenetic analysis, the isolate was taxonomically classified into the genus Dysmorphococcus, and was designated Dysmorphococcus globosus-HI (Himalayan, India). The phylogenetic analysis was performed on the basis of blast results (Figure 3) for the maximum likelihood method and involved 16 nucleotide sequences using MEGA X software [54]. All ambiguous positions were removed for each sequence pair (pairwise deletion option) and a total of 2879 positions in the final dataset were observed. The resultant tree using the 18S rDNA genes revealed that the newly isolated microalga strain has a maximum DNA sequence similarity with Dysmorphococcus globosus strain SAG 20-1 (KM020136.1) and D. globosus (X91629.1) (Figure 3). Based on the phylogenetic analysis, the isolate was taxonomically classified into the genus Dysmorphococcus, and was designated Dysmorphococcus globosus-HI (Himalayan, India).

Growth Study in Different Media
The isolated algal strain (D. globosus-HI) was cultivated under 7 different culture media to identify the best growth medium and improve its production of biochemical components. The growth curve under different culture media of D. globosus-HI is presented in Figure 4. The cultures reached a stationary phase in about 22-25 days under the tested media, except in the case of JW where the stationary phase was observed around the 20th day. During the stationary phase, the color of the cells gradually changed from green to yellowish, and thereafter reddish-orange. After day 12, the highest OD was observed in MBBM medium followed by CM and OHM medium. The lowest OD values were observed in BG-11 and JW medium after ~14th day (Figure 4). In the JW medium, the highest

Growth Study in Different Media
The isolated algal strain (D. globosus-HI) was cultivated under 7 different culture media to identify the best growth medium and improve its production of biochemical components. The growth curve under different culture media of D. globosus-HI is presented in Figure 4. The cultures reached a stationary phase in about 22-25 days under the tested media, except in the case of JW where the stationary phase was observed around the 20th day. During the stationary phase, the color of the cells gradually changed from green to yellowish, and thereafter reddish-orange. After day 12, the highest OD was observed in MBBM medium followed by CM and OHM medium. The lowest OD values were observed in BG-11 and JW medium after~14th day (Figure 4). In the JW medium, the highest growth was observed at the beginning (days [8][9][10][11][12][13][14], growth was then stabilized (days [14][15][16][17][18][19][20], and after started declining (days 24-28).
The growth characteristics of the D. globosus-HI were significantly (p ≤ 0.05) affected by the different culture media, the MBBM media (followed by CM and OHM) promoted the growth to the maximum, while BG-11 showed an opposite trend ( Table 1). The effect of the different culture media on the growth was observed in the descending order of MBBM > CM > OHM > BBM > 3N-BBM > JW > BG-11 (Table 1). The MBBM media showed significantly higher biomass yield (1.14 g L −1 ), productivity (45.82 mg L −1 d −1 ), specific growth rate (0.08 day −1 ), division per day (0.12), and maximum cellular yield (6.16 × 10 6 ) compared to the other tested media. In contrast, MBBM, CM, and OHM attained the lowest doubling time (hours). However, the lowest biomass yield (0.56 g L −1 ), productivity (22.60 mg L −1 d −1 ), maximum cellular yield (3.18 × 10 6 ), specific growth rate (0.06 day −1 ), and division per day (0.08) was recorded in the BG-11 medium with the highest doubling time (12.03 h).   Table S1. The optical density was measured at an interval of two days.
The growth characteristics of the D. globosus-HI were significantly (p ≤ 0.05) affected by the different culture media, the MBBM media (followed by CM and OHM) promoted the growth to the maximum, while BG-11 showed an opposite trend ( Table 1). The effect of the different culture media on the growth was observed in the descending order of MBBM > CM > OHM > BBM > 3N-BBM > JW > BG-11 (Table 1). The MBBM media showed significantly higher biomass yield (1.14 g L −1 ), productivity (45.82 mg L −1 d −1 ), specific growth rate (0.08 day −1 ), division per day (0.12), and maximum cellular yield (6.16 × 10 6 ) compared to the other tested media. In contrast, MBBM, CM, and OHM attained the lowest doubling time (hours). However, the lowest biomass yield (0.56 g L −1 ), productivity (22.60 mg L −1 d −1 ), maximum cellular yield (3.18 × 10 6 ), specific growth rate (0.06 day −1 ), and division per day (0.08) was recorded in the BG-11 medium with the highest doubling time (12.03 h).

Chlorophyll a, Chlorophyll b, and Total Carotenoids Content
Among all of the 7 tested culture media, chlorophyll a was observed higher than chlorophyll b and total carotenoids ( Figure 5). D. globosus-HI had a significantly higher chlorophyll a and b content in the MBBM (2.97: 1.78 µg mL −1 ), followed by CM (2.08: 1.41 µg mL −1 ), and OHM (1.51: 1.23 µg mL −1 ), respectively. In contrast, the lowest chlorophyll a and b contents (0.75: 0.62 µg mL −1 ) respectively were observed in the BG-11 medium. For the total carotenoids, the highest value was observed in MBBM medium (1. 25 µg mL −1 ), followed by 3N-BBM (1.02 µg mL −1 ) and CM (0.97 µg mL −1 ). However, the lowest total carotenoid content was observed in OHM medium (0.50 µg mL −1 ). Algal samples were harvested on day 25 of the cultivation. Values are the mean ± SD (n = 3). Values with similar letters are non-significantly different at p ≤ 0.05 using Tukey's test. Letters apply only within the same parameter. For the details of the used media, see Table S1.

Chlorophyll a, Chlorophyll b, and Total Carotenoids Content
Among all of the 7 tested culture media, chlorophyll a was observed higher than chlorophyll b and total carotenoids ( Figure 5). D. globosus-HI had a significantly higher chlorophyll a and b content in the MBBM (2.97: 1.78 μg mL −1 ), followed by CM (2.08: 1.41 μg mL −1 ), and OHM (1.51: 1.23 μg mL −1 ), respectively. In contrast, the lowest chlorophyll a and b contents (0.75: 0.62 μg mL −1 ) respectively were observed in the BG-11 medium. For the total carotenoids, the highest value was observed in MBBM medium (1. 25 μg mL −1 ), followed by 3N-BBM (1.02 μg mL −1 ) and CM (0.97 μg mL −1 ). However, the lowest total carotenoid content was observed in OHM medium (0.50 μg mL −1 ).  Table S1. Bars with similar letters are non-significantly different at p ≤ 0.05 using Tukey's test. Letters apply only within the same parameter.

Thin Layer Chromatography of Total Pigment Extracts
The total pigment extracts of D. globosus-HI from different media were spotted on the TLC plates as presented in Figure 6. These were compared with ASX control reference, i.e., the lower band represented the free ASX and β-carotene represented by the upper band. The separation of the different carotenoid pigments on the TLC plate indicated the presence of free ASX, β-carotene, and chlorophylls under different tested media. According to the RFs values, the free ASX showed an RFs value of 0.35, while the astaxanthin monoesters and diesters bands showed RFs values ranging from 0.55 to 0.725 and RFs 1.0 for β-carotene. Below the free ASX bands, several bands ranging from a deep to light green color indicated the presence of pheophytins and chlorophyll pigments.  Table S1. Bars with similar letters are non-significantly different at p ≤ 0.05 using Tukey's test. Letters apply only within the same parameter.

Thin Layer Chromatography of Total Pigment Extracts
The total pigment extracts of D. globosus-HI from different media were spotted on the TLC plates as presented in Figure 6. These were compared with ASX control reference, i.e., the lower band represented the free ASX and β-carotene represented by the upper band. The separation of the different carotenoid pigments on the TLC plate indicated the presence of free ASX, β-carotene, and chlorophylls under different tested media. According to the RFs values, the free ASX showed an RFs value of 0.35, while the astaxanthin monoesters and diesters bands showed RFs values ranging from 0.55 to 0.725 and RFs 1.0 for β-carotene. Below the free ASX bands, several bands ranging from a deep to light green color indicated the presence of pheophytins and chlorophyll pigments. Biology 2022, 11, x FOR PEER REVIEW 12 of 24

Proteins and Carbohydrates' Content
The 3N-BBM and MBBM were the most promising media for protein and carbohydrate production (Figure 7). The D. globosus-HI accumulated a significantly higher quantity of proteins in MBBM medium (122. 78

Proteins and Carbohydrates' Content
The 3N-BBM and MBBM were the most promising media for protein and carbohydrate production ( Figure 7). The D. globosus-HI accumulated a significantly higher quantity of proteins in MBBM medium (122. 78

Proteins and Carbohydrates' Content
The 3N-BBM and MBBM were the most promising media for protein and carbohydrate production (Figure 7). The D. globosus-HI accumulated a significantly higher quantity of proteins in MBBM medium (122. 78 Table S1.

Astaxanthin Analysis by HPLC
The D. globosus-HI cells extract showed a peak at the same retention time as the standard ASX, confirming the presence of ASX ( Figure S2). ASX was expressed using three different means, (i) ASX concentration (mg L −1 ), (ii) ASX content (% CDW) and (iii) ASX productivity (mg L −1 d −1 ) for D. globosus-HI tested using 7 different culture media are presented in Figure 10A,B and Table S4. D. globosus-HI was able to produce ASX in all media but at different levels. The highest ASX concentration and productivity of D. globosus-HI were attained in the 3N-BBM medium, followed by JW and MBBM ( Figure 10A,B and Table S4). The lowest ASX concentration was recorded for OHM and CM media.

Astaxanthin Analysis by HPLC
The D. globosus-HI cells extract showed a peak at the same retention time as the standard ASX, confirming the presence of ASX ( Figure S2). ASX was expressed using three different means, (i) ASX concentration (mg L −1 ), (ii) ASX content (% CDW) and (iii) ASX productivity (mg L −1 d −1 ) for D. globosus-HI tested using 7 different culture media are presented in Figure 10A,B and Table S4. D. globosus-HI was able to produce ASX in all media but at different levels. The highest ASX concentration and productivity of D. globosus-HI were attained in the 3N-BBM medium, followed by JW and MBBM ( Figure 10A,B and Table S4). The lowest ASX concentration was recorded for OHM and CM media.

Morphological Study
The new isolate has 93.39% 18S rDNA sequence similarity with previously sequenced Dysmorphococcus sp. strains (Dysmorphococcus globosus strain SAG 20-1 (KM020136.1) and D. globosus (X91629.1) in the NCBI database; therefore, it was named Dysmorphococcus globosus-HI (HI stands for Himalayan India), accession number H210309. The spherical cells of D. globosus-HI formed large clusters of aplanospores and dividing cells. Each cell had an urn or parietal-shaped chloroplast, typically 1-2 pyrenoids, with two equal "isokontic" flagella as reported by Bold and Starr [78] for Dysmorphococcus globosus strain SAG 20-1 and Dawson and Harris [79] for D. globosus (X91629.1). The D. globosus has a spherical cell up to 28 μm in diameter [78,79]. However, in the present study, the cells of D. globosus-HI grown under laboratory conditions were measured 100-200 μm in diameter, which could be due to different geographical and climate conditions and the ability to accumulate more amount of carotenoids.
Regarding asexual reproduction, the cells enlarge, settle to the bottom, and withdraw their flagella. The pyrenoids and nucleus are then divided, followed by the division of the entire protoplast, resulting in four daughters' cells surrounded by the parental cell wall.  Table S1. Bars with similar letters are non-significantly different at p ≤ 0.05 using Tukey's test. Letters apply only within the same parameter.

Morphological Study
The new isolate has 93.39% 18S rDNA sequence similarity with previously sequenced Dysmorphococcus sp. strains (Dysmorphococcus globosus strain SAG 20-1 (KM020136.1) and D. globosus (X91629.1) in the NCBI database; therefore, it was named Dysmorphococcus globosus-HI (HI stands for Himalayan India), accession number H210309. The spherical cells of D. globosus-HI formed large clusters of aplanospores and dividing cells. Each cell had an urn or parietal-shaped chloroplast, typically 1-2 pyrenoids, with two equal "isokontic" flagella as reported by Bold and Starr [78] for Dysmorphococcus globosus strain SAG 20-1 and Dawson and Harris [79] for D. globosus (X91629.1). The D. globosus has a spherical cell up to 28 µm in diameter [78,79]. However, in the present study, the cells of D. globosus-HI grown under laboratory conditions were measured 100-200 µm in diameter, which could be due to different geographical and climate conditions and the ability to accumulate more amount of carotenoids.
Regarding asexual reproduction, the cells enlarge, settle to the bottom, and withdraw their flagella. The pyrenoids and nucleus are then divided, followed by the division of the entire protoplast, resulting in four daughters' cells surrounded by the parental cell wall. These observations of the asexual reproduction for our isolate were similar to those described by Bold and Starr [78], and they also observed 4-16 daughter cells from each mother cell. After the separation of the daughter cells, they grew to the same size as the mother cell and form a spherical, dark aplanospores as mature cells. We have not observed any sexual reproduction during our study as was reported for Dysmorphococcus genus [78]. Therefore, we need more focused studies regarding this aspect. Dawson and Harris [79] stated that the sexual reproduction of Dysmorphococcus genus is isogamous to anisogamous, with the clones being homothallic.
The genus Dysmorphococcus belongs to the family Phacotaceae within the order of the Chlamydomonadales, class Chlorophyceae [80] based on the Algae Base for taxonomy (https://www.algaebase.org/search/species/detail/?species_id=59395) (accessed on 3 April 2022). However, only 5 species of this genus isolated from Asia, Europe, and North America are known, i.e., D. globosus [78], D. variabilis [80], D. sarmaii [81], D. coccifer [82], and D. punctatus [83]. Among these species, only D. variabilis and D. sarmaii have been isolated from India. This is the first study that successfully identified the D. globosus isolate from India and quantification of its biochemical compounds was estimated.
The change in the color of the culture media (from green to red) indicates the aging of the culture and pigment accumulation. Therefore, the growth of the D. globosus-HI was distinguished based on cells' age, i.e., the green cells were termed young cells, and reddish-orange-colored cells were called old cells. There was a gradual change in the color of the culture media until the entire cytoplasm became orange-red, a phenomenon similarly reported for H. pluvialis, known for ASX production. When the orange-red culture was inoculated into a fresh medium, the newly produced flagellated cells were transformed into non-motile cells within 5 days. All these morphological changes are consistent with the typical characteristics of D. globosus reported by Dawson and Harris [79] and by Neofotis et al. [84] Thus, based on common findings, this isolate was designated as D. globosus-HI, which has potential for astaxanthin production.

Assessment of an Appropriate Culture Medium
D. globosus-HI was studied in 7 different media in order to identify the best media for the growth and production of a higher amount of biochemical compounds. The growth characteristics of D. globosus-HI were studied along with the determination of carbohydrates, protein, lipid, FAs, and ASX contents. The result showed that the isolate could thrive in a broad range of media. However, MBBM medium was observed to be best for supporting its highest growth in terms of maximum OD, CDW, cell numbers, and biomass productivity, whereas BG-11 medium was recorded with the least OD and CDW, cell number, and biomass productivity. The higher biomass under the MBBM medium was consistent with previously published reports for other strains [19,50,85]. These findings might be attributed to the pH buffer system (K 2 HPO 4 -KH 2 PO 4 ), a sufficient N supply, and the higher orthophosphate concentration in the MBBM medium compared with other media [50]. The highest final biomass concentration (1.14 gL −1 ) was achieved when cells were harvested on day 25. This result indicated that, compared to several other algal strains, the growth of our isolate could be a challenge, and low biomass production could limit its ability to produce reasonable amounts of bioproducts for commercial requirements. For example, the highest growth obtained under our conditions amounted only to 1.14 g L −1 , whereas the growth of the well-known ASX source strains (H. pluvialis) amounted to 9.00 g L −1 in 15 days [19].

Lipids and FAs Profile
Using the D. globosus-HI, the highest growth was recorded in the MBBM, and the highest lipid content, lipid percentage, and productivity was recorded in 3N-BBM (32.51 mg L −1 , 6.50%, 2.60 mg g −1 d −1 ) followed by MBBM (19.69 mg L −1 , 5.58%, 2.43 mg g −1 d −1 ). Thus, 3N-BBM medium showed a balanced accumulation of biomass and lipids and attained the highest lipid productivity. It is established that microalgae is a biofuel source, since their lipid content usually ranges from 20 to 50% of the cell dry weight, and can increase to the tune of 80% under specific stress conditions [98,99]. Several algal strains, i.e., Chlorella and Haematococcus, have been proposed as potential biofuel sources due to their high lipid content, fatty acid composition, and biomass [84,100,101]. However, based on the obtained lipid content and productivity, D. globosus-HI stain may not be a potential biofuel source compared to lipid-rich strains. Besides the low lipid content and productivity of the isolate, the FAs profile revealed a high percentage of SFAs (ranging from 42.01-64.12%), indicating poor cold flow properties of the produced biodiesel, because SAFs crystallize at high temperatures and are associated with a high melting point. Therefore crystals are formed at low temperatures [100,102], negatively affecting the quality of the produced biodiesel owing to its unsuitable viscosities and poor cold-flow properties [103].

Astaxanthin Analysis
The HPLC analysis revealed that D. globosus-HI accumulates the ASX. The D. globosus isolated from North America by Neofotis et al. [84], was not quantified for the ASX content or productivity. In the present study, the ASX varied in different culture media from 37.00 to 391.00 mg L −1 , while the ASX productivity ranged from 1.48 to 15.64 mg L −1 d −1 . Changes in ASX content and productivity due to changes in the growth conditions were reported in other strains [19,100]. For example, Wang et al. [19] revealed that the ASX content varied from 1.60% to 2.70% by changing the growth conditions of H. pluvialis-JNU35. Despite the MBBM medium producing the highest biomass (1.14 g L −1 ) than the other tested media, the high biomass came at the cost of the ASX content and productivity, where the highest ASX content (391.00 mg L −1 ) and highest ASX productivity (15.60 mg L −1 d −1 ) was attained in the 3N-BBM medium, where the biomass produced only 0.74 g L −1 . Similar results were obtained by Wang et al. [19] who reported the highest ASX content (2.40%) but not the highest biomass. In the case of 3N-BBM medium, despite a relatively lower growth of D.
globosus-HI, the highest ASX productivity was recorded (in 3N-BBM medium) due to the high ASX content, confirming that the high ASX content is vital for attaining the highest ASX productivity. The H. pluvialis (standard ASX source) can accumulate lipids under stress conditions, which increases the cell size and improves ASX content, as lipids can serve as a supportive matrix, allowing for ASX pending in the cytoplasm [19,32,108]. Several authors revealed that βcarotene converts to ASX in lipid bodies and ASX accumulation is proportional to lipid synthesis [109][110][111]. Our results supported this concept through 3N-BBM medium, which showed the highest ASX content and productivity, along with the highest lipid content and productivity. Moreover, for the 7 tested media, a strong positive correlation has been observed between lipids-and ASX productivity in which the r value was 0.94, R 2 was 0.88 and the correlation has been described by a third-degree a polynomial equation ( Figure S3). Chen et al. [112] studied the correlation between ASX-and lipids accumulation and reported that an ASX esterification step might be responsible for this correlation. The authors indicated that increasing the titer of lipid content might be able to increase the ASX and that overexpression of relevant esterifying enzymes might be able to promote both lipid and ASX synthesis [112]. Under stress conditions (N-starvation), ASXproducing strains, i.e., Haematococcus, could divert C-partition from starch to lipids and ASX via increased activity of the tricarboxylic acid cycle [113]. The ASX accumulation could be induced by nutrient deficiency, especially N-deprivation. One evidence is that the cells reach the stationary phase (cells became orange-red indicating ASX accumulation) without the replacement of a new nutrient medium and when orange-red cells were inoculated into a fresh medium, the newly produced cells completely turn green within 5 days.
In our study, the highest ASX content (391.00 mg L −1 ) observed in D. globosus-HI was higher than any known potential algal ASX source, where the genus Haematococcus has an ASX content ranging from 7.72 to 174.70 mg L −1 [19,50,85,100,114]. Similarly, the ASX percentage (CDW basis) in our isolate (51.01% CDW) was also higher than any observed values for Haematococcus (1.00 to 5.00%) and Bracteacoccus aggregatus BM5/15 (48.01%) [74]. More importantly, despite the low biomass of D. globosus-HI, ASX productivity (15.00 mg L −1 d −1 ) was promising, which is higher than any other algal strains [50,85,100,114]. The only exception is H. pluvialis JNU35, only when cultivated at a photobioreactor scale under specific stress conditions (different N-sources and light conditions) [19].
Our results indicate that D. globosus-HI has excellent genetic properties with a higher accumulation of ASX. D. globosus-HI could provide a comparable ASX productivity to Heamatococcus sp., despite its low biomass productivity. As observed for Heamatococcus sp. [19,32,50,115], subjecting D. globosus-HI to specific growth conditions, i.e., nutrient deprivation/enrichment, salinity, irradiance, and temperature, could further enhance its growth, ASX content, and productivity. The D. globosus-HI strain produced low biomass; therefore, it requires further media engineering to enhance its growth. In particular, C-enrichment could be a win-win solution that might provide high growth without decreasing the ASX accumulation. Several authors revealed that a higher biomass and ASX yield could be achieved by altering the C/N ratio through C-enrichment [109,[116][117][118]. In our study, ASX was determined during the early stationary phase; however, ASX content and productivity could differ if measured during the mid-and/or the late stationary phase. Future research is required to determine the best harvesting time to get the highest ASX productivity. Therefore, in the future, work should be focused on enhancing the growth of D. globosus-HI without affecting its ASX content through the optimization of physio-chemical environmental conditions (C-enrichment and illumination) and/or genetic modification.

Conclusions
A new microalgal strain was successfully isolated from the Himalayan region, Northern India. Based on the morphological and molecular analysis, the isolated strain was identified as D. globosus-HI. This is the first report that quantifies its potential to produce value-added bioproducts and highlights its great potential as a new source of natural ASX.